Technical Abstract:
Mechanical wounding, one of the first steps in both pathogen infection and herbivore attack, activates signal transduction pathways dedicated to defense and recovery. The signaling pathways include reversible protein phosphorylation, changes in intracellular calcium levels, alarmone production, and transcriptional gene activation. Protein phosphorylation modulates activity, transmitting signals within cellular pathways and networks. Mechanical wounding of maize (Zea mays) leaves results in altered activity of the ZmCPK11 CDPK. Protein kinase activity was monitored using in-gel kinase assays. Activity increased 15 min after cutting the leaves, then slowly declined. By 20 h after wounding, ZmCPK11 activity had returned to the basal level. We determined that ZmCPK11 homologs are present only in monocots, then used barley (Hordeum vulgare) EST sequences to isolate the corresponding CDPK, HvCDPK12. The levels of HvCDPK12 activity increased 15 min after leaf wounding similar to the ZmCPK11-pattern, suggesting that it is both a structural and functional homolog. Rapid transient activation of these CDPKs indicates an involvement in the early stages of stress signal transduction. After the events that occur within the first min of wounding (production and perception of primary signals, activation of ion channels at the plasma membrane, and reversible protein phosphorylation), mechanisms are activated to generate a second wave of wound-related signals likely directed to either propagate defense responses or activate systemic defense functions. Among the secondary signals are oxylipin alarmones including jasmonic acid (JA). There is also an alternative, JA-independent pathway leading to transcriptional gene activation. Both pathways are regulated by reversible protein phosphorylation. In 14 d-old maize plants there is increased ZmCPK11 activity 60 min after treatment with MeJA, to a maximum activity after 30 min. This suggests that ZmCPK11 is involved in the JA-dependent pathway. To better understand stress-induced signal transduction, we have begun to analyze changes in the maize leaf phospho-proteome in response to mechanical injury. Multiplex-staining of high resolution 2D gels for protein (Sypro Ruby) and phosphorylation (Pro-Q Diamond) allows us to quantify changes in protein phosphorylation status after wounding. The detected maize P-proteins were identified by Mass Spectrometry. We now hope to identify specific kinase clients and to unravel the dynamics of information flow through the wound-induced signaling pathways.